尼可地尔对抗高糖引起的H9c2心肌细胞损伤和炎症反应

Nicorandil protects H9c2 cardiac cells against high glucose-induced injury and inflammation

目的探讨尼可地尔(nicorandil,Nic)能否通过调控核因子-κB(nuclear factorκB,NF-κB)/环氧化酶-2(cyclooxygenase-2,COX-2)通路对抗高糖引起的H9c2心肌细胞损伤和炎症。方法应用细胞计数盒(CCK-8)检测心肌细胞存活率;蛋白质免疫印迹法测定NF-κB、COX-2和cleaved caspase-3蛋白的表达水平;乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒检测细胞培养液中LDH的活性;双氯荧光素染色荧光显微镜照相法测定胞内活性氧(reactive oxygen species,ROS)水平;Hoechst 33258核染色荧光显微镜照相法测定凋亡细胞数量;罗丹明123染色荧光显微镜照相法检测线粒体膜电位(mitochondrial membrane potential,MMP);ELISA法检测细胞培养液中白介素^(-1)β(IL^(-1)β)和肿瘤坏死因子-α(TNF-α)的水平。结果 35 mmol·L^(-1)葡萄糖(高糖,HG)作用H9c2心肌细胞24 h能明显降低心肌细胞存活率。在HG作用前,应用20~100μmol·L^(-1)Nic预处理60 min或50μmol·L^(-1)Nic预处理30~120 min均可明显对抗HG对心肌细胞存活率的抑制作用。另一方面,HG可上调心肌细胞磷酸化(p)-NF-κB p65和COX-2的表达,50μmol·L^(-1)Nic预处理心肌细胞60 min能抑制HG诱导的p-NF-κB p65和COX-2表达的上调。此外,HG作用心肌细胞24 h可引起明显的心肌细胞损伤和炎症反应,使培养液中LDH活性、ROS生成、凋亡细胞数量、cleaved caspase-3表达、MMP丢失及炎症因子IL^(-1)β和TNF-α的分泌均增加;在HG作用前,应用50μmol·L^(-1)Nic预处理心肌细胞60 min或应用100μmol·L^(-1)PDTC(NF-κB抑制剂)或10μmol·L^(-1)NS-398(COX-2抑制剂)和HG共处理心肌细胞24 h能明显拮抗HG引起的上述损伤和炎症反应。结论 Nic通过抑制NF-κB/COX-2通路对抗HG引起的H9c2心肌细胞损伤和炎症。

Aim To investigate whether nicorandil（ Nic） protects H9c2 cardiac cells against high glucose（ HG）-induced injury and inflammation by inhibiting nuclear factor-κB（ NF-κB） / cyclooxygenase-2（ COX-2） pathway. Methods Cell viability was measured by cell counter kit-8（ CCK-8） assay. The expression levels of NF-κB,COX-2 and cleaved caspase-3 were determined by Western blot. The activity of lactate dehydrogenase（ LDH） in the culture medium was measured with commercial kits. The intracellular level of reactive oxygen species（ ROS） was detected by 2＇,7＇-dichlor-fluorescein-diacetate（ DCFH-DA） staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. Mitochondrial membrane potential（ MMP） was examined by rhodamine 123 staining followed by photofluorography. The secretion levels of interleukin-1β（ IL-1β） and tumor necrosis factor-α（ TNF-α） were detected by ELISA. Results After H9c2 cardiac cells were treated with 35 mmol · L^-1 glucose（ high glucose,HG） for 24 h,the cell viability was significantly decreased. Pre-treatment of the cellswith 20 - 100 μmol·L^-1 Nic for 60 min or 50 μmol·L^-1 Nic for 30 -120 min before exposure to HG significantly attenuated the decrease in viability induced by HG. On the other hand,HG increased the expression levels of phosphorated（ p）-NF-κB p65 and cyclooxygenase-2（ COX-2） in H9c2 cardiac cells. Pre-treatment of the cells with 50 μmol·L^-1 Nic for 60 min attenuated the up-regulation of p-NF-κB p65 and COX-2expression levels induced by HG. Furthermore,HG induced considerable injuries and inflammatory response,leading to increases in LDH activity, ROS generation,MMP loss,the number of apoptotic cells,the expression of cleaved caspase-3 as well as the se-cretion levels of IL-1β and TNF-α. Pre-treatment of the cells with 50 μmol·L^-1Nic for 60 min before HG exposure,or co-treatment of the cells with 100 μmol·L^-1PDTC（ an inhibitor of NF-κB） or 10 μmol·L^-1NS-398（ an inhibitor of COX-2） and HG for 24 h obviously reduced the above injuries and inflammatory response induced by HG. Conclusion Nic protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting NF-κB / COX-2 pathway.